N-methyl Amino Acids Research Articles

Validation of the Distal Effect of Electron-Withdrawing Groups on the Stability of Peptide Enolates and Its Exploitation in the Controlled Stereochemical Inversion of Amino Acid Derivatives

Theoretical studies had predicted that N-electron-withdrawing substituents, hydrogen bonding, and protonation at amide nitrogen selectively increase the acidity of a distal proton adjacent to the amide carbonyl to the extent that the α-carbonyl acidity of some N-substituted amides exceeds that of typical ketones. Now, in the present work, competitive, base-catalyzed hydrogen-deuterium exchange experiments, with diisopropyl ketone and a series of N-substituted acetamides and diketopiperazines, have established that there is a strong correlation between the calculated acidities and the experimental rates of deprotonation in these systems. The results show that the rates of exchange of the distal protons of N-acylated and N-sulfonylated amides are more than 4 orders of magnitude faster than those of the N-methylated analogues and that the acylated and sulfonylated amides are much more reactive in this regard than diisopropyl ketone. The magnitude and regioselectivity of the distal effect is sufficient for practical applications and has been exploited in the manipulation of N-acetyl α,α'-disubstituted diketopiperazines for the controlled α-deuteration and stereochemical inversion of N-methylamino acids, and in the production of α-deuterated (2R,3S)-N-methylalloisoleucine from the nondeuterated (2S,3S)-isoleucine diastereomer.

Flexizymes for genetic code reprogramming

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions. Preparation of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in approximately 1 week. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.

Verrucamides A–D, Antibacterial Cyclopeptides from Myrothecium verrucaria

Four new cyclic tetradecapeptides, verrucamides A-D (1-4), have been isolated from the solid-substrate fermentation culture of the ascomycete fungus Myrothecium verrucaria. The structures of these compounds, each featuring six N-methylated amino acid residues, were elucidated primarily by NMR and MS methods. The absolute configurations of 1-4 were assigned by application of Marfey's method on their acid hydrolysates. Compounds 1-4 showed antimicrobial activity against the Gram-positive bacterium Staphylococcus aureus.

The Combination of Prolinoamino Acids and Cyclopropylamino Acids Leads to Fully Functionalized, Stable β‐Turns in Water

Tight turns: Peptide mimics based on combinations of cis-3-prolinoamino acids (prolinohomotryptophan) and (Z)-2,3-methanoamino acids (c3Arg or c3Lys) form stable β-turns in water. This combination of constraints allows one to mimic turns with side chain conformations not accessible with the previously reported strategy involving combinations of prolinoamino acids and N-methylamino acids. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Veraguamides A−G, Cyclic Hexadepsipeptides from a Dolastatin 16-Producing Cyanobacterium Symploca cf. hydnoides from Guam

Cytotoxicity-directed purification of a Symploca cf. hydnoides sample from Cetti Bay, Guam, afforded seven new cyclic depsipeptides, veraguamides A-G (1-7), together with the known compound dolastatin 16. The planar structures of 1-7 were elucidated using NMR and MS experiments, while enantioselective HPLC and Mosher's analysis of acid and base hydrolysates, respectively, were utilized to assign the absolute configurations of the stereocenters. Veraguamides A-G (1-7) are characterized by the presence of an invariant proline residue, multiple N-methylated amino acids, an α-hydroxy acid, and a C8-polyketide-derived β-hydroxy acid moiety with a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These compounds and a semisynthetic analogue (8) showed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure-activity relationship analysis identified several sensitive positions in the veraguamide scaffold that affect the cytotoxic activity of this compound class. Dolastatin 16 showed only weak cytotoxic activity on both cell lines tested. The complete stereostructure of dolastatin 16 was proposed for the first time through degradation followed by a combination of advanced Marfey's analysis and modified Mosher's analysis using phenylglycine methyl ester as a chiral anisotropic reagent.

Optimal codon choice can improve the efficiency and fidelity of N-methyl amino acid incorporation into peptides by in-vitro translation.

premature termination of translation can occur or incorrect amino acids can be misincorporated in place of the desired NMe AA, resulting in reduced incorporation efficiency and fidelity of the N-Me AA. We sought to determine the main cause of such misincorporation and truncation events, and then to optimize the incorporation of N-Me AAs. We examined the efficiency and fidelity with which three different N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) are incorporated at a single position in a short peptide (MH6MXmEP, Xm = N-Me AA, M = Met, E = Glu, P = Pro, H = His) using each of the codons of the corresponding natural amino acid (Figure 1). We showed previously that these three N-Me AAs can be incorporated into peptides by in-vitro translation [11] using the fully reconstituted PURE system for translation (PURE = protein synthesis using recombinant elements, an in vitro system containing only purifies components). [13] We directed the incorporation of these N-Me AAs into peptides by supplying in-vitro translation reaction mixtures with total tRNA that had been enzymatically precharged with Leu, Thr, or Val and then chemically N-methylated as previously described (see Experimental Section). [7, 11] The presence of the other 19 amino acid/ aminoacyl-tRNA synthetase (AARS) pairs in the translation

Deletion of Ac-NMePhe(1) from [NMePhe(1) ]arodyn under acidic conditions, part 1: effects of cleavage conditions and N-terminal functionality.

Peptides containing N-methylamino acids can exhibit improved pharmacodynamic and pharmacokinetic profiles compared to nonmethylated peptides, and therefore interest in these N-methylated peptides has been increasing in recent years. Arodyn (Ac[Phe¹,²,³,Arg⁴,D-Ala⁸]Dyn A(1-11)NH₂) is an acetylated dynorphin A(Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al., J Med Chem 2002, 45, 5617), and its analog [NMePhe¹]arodyn shows even higher affinity and selectivity for κ opioid receptors (Bennett et al., J Pept Res 2005, 65, 322). During the synthesis of [NMePhe¹]arodyn analogs, the arodyn-(2-11) derivatives were obtained as major products. Analysis indicated that Ac-NMePhe was lost from the completed peptide sequence during acidic cleavage of the peptides from the resin and that the acetyl group played an important role in this side reaction. Different cleavage conditions were evaluated to minimize this side reaction and maximize the yield of pure [NMePhe¹]arodyn analogs. Modifications to the N-terminus of the peptides to prevent the side reaction were also explored. The incorporation of a heteroatom-containing group such as methoxycarbonyl as the N-terminal functionality prevented this side reaction, while the incorporation of a bulky acyl group could not. Substituting NMePhe with the conformationally constrained analog Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) also prevented the side reaction.

SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

Cloning and Expression Analysis of Cyclic Peptide Synthetase from Fusarium oxysporum KFCC11363P

Fusarium oxysporum KFCC11363P produces four different cyclohexadepsipeptides. A cyclicpeptide synthetase gene from F. Oxysporum KFCC11363P, FoCPS1, was cloned and analyzed. The open reading frame of FoCPS1 consisted of 9486 bp without an intron, and the predicted protein encoded was comprised of 3162 amino acids. FoCPS1 exists as a single copy and is not present in F. oxysporum strains that do not generate cyclohexadepsipeptides. Expression of FoCPS1 reached maximum at day 3 after inoculation and was not expressed well in the medium in which cyclicpeptides was not produced. FoCPS1 is comprised of two activation, three thiolation, three condensation, and one N-methylation domains. Based on the non-ribosomal code analysis and the domain arrangement, the first adenylation domain is likely to specify carboxylic acid derivatives such as hydroxyisovaleic acid or 2-hydroxy-3-methylpentanoic acid, and the second adenylation domain is likely to specify an N-methyl amino acid, such as N-methyl valine.

ChemInform Abstract: Oxybenzotriazole Free Peptide Coupling Reagents for N-Methylated Amino Acids.

Abstract The main product of N-methylated amino acid coupling, when oxybenzotriazole is present in the reagent (BOP, PyBOP, HBPyU) or as an additive (DCC/HOBt), is the weakly reactive benzotriazolyl ester. On the other hand, the corresponding new halogenated reagents PyBroP, PyCloP and PyClU give very good results and can be recommended.

ChemInform Abstract: A New Approach to N-Methylaspartic, N-Methylglutamic, and N-Methyl-α-aminoadipic Acid Derivatives.

N-Methylaspartic acid derivatives and its homologues are obtained by a stereoconservative one-pot procedure from hexafluoroacetone-protected aspartic and glutamic acid, 2a and 2b, respectively. α-Aminoadipic acid (5c) and its derivative 6c are accessible from the corresponding glutamic acid derivative 9b by a Wolff rearrangement. A variety of homochiral N-methylamino acids, 5 and 12, and their derivatives, 6 and 8-11, become readily available by the new synthetic concept.

ChemInform Abstract: The 1,5-Disubstituted Tetrazole Ring as a cis-Amide Bond Surrogate

Proline occupies a special role among those amino acids incorporated into peptides and proteins by the normal ribosomal pathways, since it is the only residue that leads to an N-alkyl amide bond. In peptide natural products that often have special biosynthetic pathways or unusual posttranslational modifications, N-methyl amino acids are common and may play a special role because of their conformational properties, including their proclivity for cis-trans isomerism of the amide bond. Numerous peptides with important biological activities, such as cyclosporin and didemnin, contain N-methyl amino acids. Cis-trans isomerism of the N-alkyl amide bond involving the amino group can readily be observed (1) in the NMR of proline and N-methyl amino acid-containing peptides. In the case of angiotensin and thyroliberin (TRH) analogs, the quantity of cis-isomer in aqueous solution was correlated (2) with the biological activity. This suggested that the cis-isomer might be the one bound to the receptor and responsible for the observed biological activity. Bairaktari et al. (3) have reported that the normal amide bond between an He and Lys residues in the linear peptide, bombolitin, has the cis-conformation when bound to phospholipid micelles. In protei0n crystal structures, cis-amide bond conformations are occasionally observed for the normal, nonalkylated amide bond. A cis-amide bond predisposes the peptide for a reverse turn, a so-called Type VI β-turn. Brandl and Deber (4) have proposed that cis-trans isomerism of proline residue might play a role in transduction of transmembrane proteins.

ChemInform Abstract: Solution-Phase Synthesis of a Hindered N-Methylated Tetrapeptide Using Bts-Protected Amino Acid Chlorides: Efficient Coupling and Methylation Steps Allow Purification by Extraction.

N-Benzothiazole-2-sulfonyl (Bts)-protected amino acid chlorides were used to prepare the hindered cyclosporin 8-11 tetrapeptide subunit 1. The synthesis was performed via 3a and the deprotected amines 5a, 13, and 19, including three repeated cycles involving N-methylation using iodomethane/potassium carbonate, deprotection of the Bts group, and N-acylation with a N-Bts-amino acid chloride such as 9b or 9c. Among three Bts cleavage methods compared (H3PO2/THF; NaBH4/EtOH; PhSH/K2CO3), the third gave somewhat higher overall yields. N-Acylation of 5a with the Bts-protected N-methylamino acid chloride 10b followed by deprotection was also highly efficient and could be used as an alternative route to 11. Each of the deprotected amines was isolated without chromatography using simple extraction methods to remove neutral byproducts. The tetrapeptide 1 was obtained in analytically pure form as the monohydrate.

Prolinoamino Acids as Tools to Build Bifunctionalized, Stable β‐Turns in Water

β-Turn it on: Peptides incorporating cis-3-prolinoamino acids (prolinoleucine or prolinohomotryptophane) and N-methylamino acids (NMePhe/Arg/Lys) have been synthesized to mimic stable β-turns in water (see figure). These 3-substituted prolines are valuable peptidomimetic tools for synthesizing β-turns while keeping the side chain of proteinogenic amino acids.

Siamese Depsipeptides: Constrained Bicyclic Architectures

has been designed. Cyclic depsipeptide dimers connected by a CC bond were constructed. Two kind of possible connections can be envisaged, those that share a side chain bond or those that share a backbone bond. Owing to the resulting proximity of both structurally identical cycles, they can be called Siamese depsipeptides. For Siamese depsipeptides, the hydroxy acid of the natural compound was replaced with tartaric acid and the second cycle was constructed on its additional OH and CO2H function. Both modes of connection prevent steric interaction between the two cycles, and H-bond interactions are expected to be minimal because ester bonds are not H-bond donors and are weak H-bond acceptors. [5] The two Siamese depsipeptides proposed are shown in Scheme 1. The cyclic depsipeptide sansalvamide A (SA), which is produced by a marine fungus and shows cancer cell cytotoxicity, was selected as a model. SA is composed of four amino acids (2 � Leu, Phe, Val) and one hydroxy acid (leucic acid). [6] More than 80 peptidic analogues, wherein the hydroxy acid and the amino acid constituents were substituted for d-amino acids and N-methyl amino acids with preserved or altered side chains, are already reported. Some of them exhibit greater activity and better selectivity than the natural SA. [7] However, although SA is a relatively small and structurally simple depsipeptide, an active sequence has not been disclosed to date.

Systematic Study of the Structures of Potassiated Tertiary Amino Acids: Salt Bridge Structures Dominate

The gas-phase structures of a series of potassiated tertiary amino acids have been systematically investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, ion mobility spectrometry (IMS), and computational modeling. The examined analytes comprise a set of five linear N,N-dimethyl amino acids derived from N,N-dimethyl glycine and three cyclic N-methyl amino acids including N-methyl proline. The number of methylene groups in either the alkyl chain of the linear members or in the ring of the cyclic members of the series is gradually varied. The spectra of the cyclic potassiated molecular ions are similar and well resolved, whereas the clear signals in the respective spectra of the linear analytes increasingly overlap with longer alkyl chains. Measured IRMPD spectra are compared to spectra calculated at the B3LYP/6-311++G(2d,2p) level of theory to identify the structures present in the experimental studies. On the basis of these experiments and calculations, all potassiated molecular ions of this series adopt salt bridge structures in the gas phase, involving bidentate coordination of the potassium cation to the carboxylate moiety. The assigned salt bridge structures are predicted to be the global minima on the potential energy surfaces. IMS cross-section measurements of the potassiated systems show a monotonic increase with growing system size, suggesting that the precursor ions adopt the same type of structure and comparisons between experimental and theoretical cross sections are consistent with salt bridge structures and the IRMPD results.

Rapid Optimization of a Peptide Inhibitor of Malaria Parasite Invasion by Comprehensive N-Methyl Scanning

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters.

Translation Initiation with Initiator tRNA Charged with Exotic Peptides

In the prokaryotic translation system formylmethionyl-tRNA(fMet)(CAU) acts as an exclusive initiator to yield peptides bearing formylmethionine at the N-terminus. We herein report a new methodology to initiate the translation reaction with peptidyl-tRNA(fMet)(CAU) in which the peptidyl group consists of unusual building blocks, such as D-amino acids, beta-amino acids, and N-methyl amino acids, and express peptides containing a stretch of exotic peptides at the N-terminus. Synthesis of the initiator peptidyl-tRNAs was facilitated by flexizyme, a flexible tRNA aminoacylation ribozyme, and the start codon was reprogrammed by withdrawing methionine from a reconstituted E. coli cell-free translation system to reassign the initiator from formylmethionine to exotic peptides. This represents the first demonstration for initiating the ribosomal peptide synthesis with peptides, which offers us a new tool for the synthesis of a wide variety of unusual peptides and also the mechanistic studies on the initiation and/or elongation events in translation.

Total Synthesis of Cyclosporin O: Exploring the Utility of Bsmoc-NMe- Amino Acid Fluorides and KOAt

Cyclosporin O (CyO), an immunosuppressent cyclic undecapeptide, was synthesized by convergent approach employing Bsmoc-Nmethyl amino acid fluorides and Potassium Salt of 7-Aza-1-hydroxybenzotriazole (KOAt) in solution by stepwise assembly. The couplings were found to be epimerisation free. The difficulty in the coupling of four consecutive N-methyl amino acids at position 8, 9, 10 and 11 was overcome by repeating the coupling thrice at these critical positions. All the ten protected peptide fragments of CyO starting from the dipeptide to the undecapeptide and final protected as well as CyO were isolated and fully characterized.

Synthesis of 1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc) Protected N-Methyl Amino Acids by Reduction of Bsmoc-5-Oxazolidinones and Their Use in Peptide Synthesis

The synthesis of Bsmoc-N-methyl amino acids is presented. The first step involves p-toluenesulfonic acid (TsOH) catalysed condensation of a Bsmoc-amino acid with paraformaldehyde to furnish N-Bsmoc-5-oxazolidinone under MW irradiation. This intermediate is reduced to the corresponding N-methyl amino acid using triethylsilane (Et3SiH) and trifluoroacetic acid (TFA) at r.t. The N-methyl amino acids are converted into corresponding acid fluorides using diethylaminosulfur trifluoride (DAST) and employed as coupling agents in the synthesis of dipeptides. The peptide coupling was mediated by KOAt in CH2Cl2.